Analogs of parathyroid hormone

ABSTRACT

Peptide variants of fragment (1-34) of parathyroid hormone, in which at least one of the amino acid residues at positions 7, 11, 23, 24, 27, 28, and 31 is cyclohexylalanine, or at least one of the amino acid residues at positions 3, 16, 17, 18, 19, and 34 is α-aminoisobutyric acid; or, alternatively, at least the amino acid residue at position 1 is α,β-diaminopropionic acid, the amino acid residue at position 27 is homoarginine, or the amino acid residue at position 31 is norleucine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/626,186 filed Mar. 29, 1996 now U.S. Pat No. 5,723,577 which in turnclaims priority from U.S. provisional application 60/001,105 filed Jul.13, 1995 and U.S. provisional application 60/003,305 filed Sep. 6, 1995.

BACKGROUND OF THE INVENTION

Parathyroid hormone ("PTH") is a polypeptide produced by the parathyroidglands. The mature circulating form of the hormone is comprised of 84amino acid residues. The biological action of PTH can be reproduced by apeptide NH₂ ; fragment of its N-terminus (e.g. amino acid residues 1through 34). Parathyroid hormone-related protein ("PTHrP") is a 139 to173 amino acid-protein with N-terminal homology to PTH. PTHrP sharesmany of the biological effects of PTH including binding to a commonPTH/PTHrP receptor. Tregear, et al., Endocrinol., 93:1349 (1983). PTHpeptides from many different sources, e.g., human, bovine, rat, chicken,have been characterized. Nissenson, et al., Receptor, 3:193 (1993).

PTH has been shown to both improve bone mass and quality. Dempster, etal., Endocrine Rev., 14:690 (1993); and Riggs, Amer. J. Med., 91 (Suppl.5B):37S (1991). The anabolic effect of intermittently administered PTHhas been observed in osteoporotic men and women either with or withoutconcurrent antiresorptive therapy. Slovik, et al., J. Bone Miner. Res.,1:377 (1986); Reeve, et al., Br. Med. J., 301:314 (1990); and Hesch,R-D., et al., Calcif. Tissue Int'l, 44:176 (1989).

SUMMARY OF THE INVENTION

In one aspect, the invention relates to peptide variants of PTH(1-34) ofthe following generic formula: ##STR1## wherein A₁ is Ser, Ala, or Dap;

A₃ is Ser, Thr, or Aib;

A₅ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe or p-X-Phe, in which X isOH, a halogen, or CH₃ ;

A₇ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe in which X isOH, a halogen, or CH₃ ;

A₈ is Met, Nva, Leu, Val, Ile, Cha, or Nle;

A₁₁ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe or p-X-Phe in which X isOH, a halogen, or CH₃ ;

A₁₂ is Gly or Aib;

A₁₅ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe in which Xis OH, a halogen, or CH₃ ;

A₁₆ is Ser, Asn, Ala, or Aib;

A₁₇ is Ser, Thr, or Aib;

A₁₈ is Met, Nva, Leu, Val, Ile, Nle, Cha, or Aib;

A₁₉ is Glu or Aib;

A₂₁ is Val, Cha, or Met;

A₂₃ is Trp or Cha;

A₂₄ is Leu or Cha;

A₂₇ is Lys, Aib, Leu, hArg, Gln, or Cha;

A₂₈ is Leu or Cha;

A₃₀ is Asp or Lys;

A₃₁ , is Val, Nle, Cha, or deleted;

A₃₂ is His or deleted;

A₃₃ is Asn or deleted;

A₃₄ is Phe, Tyr, Amp, Aib, or deleted;

each of R₁ and R₂ is, independently, H, C ₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,C₇₋₂₀ phenylalkyl, C₁₁₋₂₀ napthlalkyl, C₁₋₁₂ hydroxyalkyl, C₂₋₁₂hydroxyalkenyl, C₇₋₂₀ hydroxyphenylalkyl, or C₁₁₋₂₀hydroxynaphthylalkyl; or one and only one of R₁ and R₂ is COE₁ in whichE₁ is C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₇₋₂₀ phenylalkyl, C₁₁₋₂₀ napthyalkyl,C₁₋₁₂ hydroxyalkyl, C₂₋₁₂ hydroxyalkenyl, C₇₋₂₀ hydroxy-phenylalkyl, orC₁₁₋₂₀ hydroxynaphthylalkyl; and

R₃ is OH, NH₂, C₁₋₁₂ alkoxy, or NH--Y--CH₂ --Z in which Y is a C₁₋₁₂hydrocarbon moiety and Z is H, OH, CO₂ H, or CONH₂ ;

provided that (i) at least one of A₅, A₇, A₈, A₁₁, A₁₅, A₁₈, A₂₁, A₂₃,A₂₄, A₂₇, A₂₈, and A₃₁ is Cha, or at least one of A₃, A₁₂, A₁₆, A₁₇,A₁₈, A₁₉, and A₃₄ is Aib; or that (ii) at least A₁ is Dap, A₇ is β-Nal,Trp, Pal, Phe, or p-X-Phe, A₁₅ is β-Nal, Trp, Pal, Phe, or p-X-Phe, A₂₇is hArg, or A₃₁ is Nle; or a pharmaceutically acceptable salt thereof.

A subset of the compounds covered by the above formula are those inwhich at least one of A₅, A₇, A₁₁, A₁₅, A₁₈, A₂₁, A₂₃, A₂₄, A₂₇, A₂₈,and A₃₁ is Cha, For example, A₃ is Ser; A₅ is Ile; A₇ is Leu or Cha; A₈is Met, Nva, Leu, Val, Ile, or Nle; A₁₁ is Leu or Cha; A₁₂ is Gly; A₁₅is Leu or Cha; A₁₆ is Asn or Aib; A₁₇ is Ser; A₁₈ is Met or Nle; A₂₁ isVal; A₂₇ is Lys, hArg, or Cha; A₃₂ is His; A₃₁ is Val, Nle, or Cha; A₃₃is Asn; A₃₄ is Phe, Tyr, Amp, or Aib; R₁ is H; R₂ is H; and R₃ is NH₂ ;provided that at least one of A₅, A₇, A₈, A₁₁, A₁₅, A₁₈, A₂₁, A₂₃, A₂₄,A₂₇, A₂₈, and A₃₁ is Cha, or at least one of A₃, A₁₂, A₁₆, A₁₇, A₁₈,A₁₉, and A₃₄ is Aib. If desired, at least one of A₇ and A₁₁ can be Cha;or at least one of A₁₅, A₂₃, A₂₄, A₂₇, A₂₈, and A₃₁ is Cha.

In another subset, at least one of A₃, A₁₂, A₁₆, A₁₇, A₁₈, A₁₉, and A₃₄is Aib. For example, A₃ is Ser or Aib; A₅ is Ile; A₇ is Leu or Cha; A₈is Met, Nva, Leu, Val, Ile, or Nle; A₁₁ is Leu or Cha; A₁₅ is Leu orCha, A₁₆ is Asn or Aib; A₁₈ is Met, Aib, or Nle; A₂₁ is Val; A₂₇ is Lys,Aib, Leu, hArg, or Cha; A₃₁ is Val, Nle, or Cha; A₃₂ is His; A₃₃ is Asn;A₃₄ is Phe, Tyr, Amp, or Aib; R₁ is H; R₂ is H; and R₃ is NH₂ ; providedthat at least one of A₅, A₇, A₈, A₁₁, A₁₅, A₁₈, A₂₁, A₂₃, A₂₄, A₂₇, A₂₈,and A₃₁ is Cha, or at least one of A₃, A₁₂, A₁₆, A₁₇, A₁₈, A₁₉, and A₃₄is Aib. If desired, at least one of A₇ and A₁₁ can be Cha; or at leastone of A₁₅, A₂₃, A₂₄, A₂₇, A₂₈, and A₃₁ is Cha.

In a still further subset, at least one of A₅, A₇, A₈, A₁₁, A₁₅, A₁₈,A₂₁, A₂₃, A₂₄, A₂₇, A₂₈, and A₃₁ is Cha, or at least one of A₃, A₁₂,A₁₆, A₁₇, A₁₈, A₁₉, and A₃₄ is Aib. For example, A₃ is Ser or Aib; A₅ isIle; A₇ is Leu or Cha; A₈ is Met, Nva, Leu, Val, Ile, or Nle; A₁₁ is Leuor Cha; A₁₅ is Leu or Cha; A₁₆ is Asn or Aib; A₁₈ is Met, Aib, or Nle;A₂₁ is Val; A₂₇ is Lys, Aib, Leu, hArg, or Cha; A₃₁ is Val, Nle, or Cha;A₃₂ is His; A₃₃ is Asn; A₃₄ is Phe, Tye, Amp, or Aib; R₁ is H; R₂ is H;and R₃ is NH₂. If desired, at least one of A₇ and A₁₁, is Cha and atleast one of A₁₆, A₁₉, and A₃₄ is Aib; or at least one of A₂₄, A₂₈, andA₃₁ is Cha and at least one of A₁₆ and A₁₇ is Aib.

In yet another subset, at least one of A₁ is Dap, A₇ is β-Nal, Trp, Pal,Phe or p-X-Phe, A₁₃ is β-Nal, Trp, Pal, Phe, or p-X-Phe. For example, A₁is Ser, Gly, or Dap; A3 is Ser or Aib; A₈ is Met, Nva, Leu, Val, Ile, orNle; A₁₆ is Asn or Aib; A₁₈ is Met, Aib, or Nle; A₂₁ is Val; A₂₇ is Lys,Aib, Leu, hArg, or Cha; A₃₁ is Val, Nle, or Cha; A₃₂ is His; A₃₃ is Asn;A₃₄ is Phe, Tyr, Amp, or Aib; R₁ is H; R₂ is H; and R₃ is NH₂.

The following are examples of the peptide of this invention as coveredby the above formula: [Cha⁷ ]hPTH (1-34)NH₂ ; [Cha¹¹ ]hPTH(1-34)NH₂ ;[Cha¹⁵ ]hPTH (1-34)NH₂ ; [Cha⁷, 11 ]hPTH(1-34)NH₂ ; [Cha⁷, 11, Nle⁸, 18,Tyr³⁴ ]hPTH (1-34)NH₂ ; [Cha²³ ]hPTH(1-34)NH₂ ; [Cha²⁴ ]hPTH(1-34)NH₂ ;[Nle⁸, 18, Cha²⁷ ]hPTH(1-34)NH₂ ; [Cha²⁸ ]hPTH(1-34)NH₂ ; [Cha³¹]hPTH(1-34)NH₂ ; [Cha²⁷ ]hPTH(1-34)NH₂ ; [Cha²⁷, 29 ]hPTH(1-34)NH₂ ;[Cha²⁸ ]bPTH(1-34)NH₂ ; [Cha²⁸ ]rPTH(1-34)NH₂ ; [Cha²⁴, 28, 31]hPTH(1-34)NH₂ ; [Aib₁₆ ]hPTH(1-34)NH₂ ; [Aib¹⁹ ]hPTH(1-34)NH₂ ; [Aib³⁴]hPTH(1-34)NH₂ ; [Aib¹⁶, 19 ]hPTH(1-34)NH₂ ; [Aib¹⁶, 19, 34]bPTH(1-34)NH₂ ; [Aib¹⁶, 34 ]hPTH(1-34)NH₂ ; [Aib¹⁹, 34 ]hPTH(1-34)NH₂ ;[Cha⁷, 11, Nle⁸, 18, Aib¹⁶, 19, Tyr³⁴ ]hPTH (1-34)NH₂ ; [Cha⁷, 11, Nle⁸,18, 31, Aib¹⁶, 19, Tyr³⁴ ]hPTH(1-34)NH₂ ; [Cha⁷, Aib¹⁶ ]hPTH(1-34)NH₂ ;[Cha¹¹, Aib¹⁶ ]hPTH(1-34)₂ ; [Cha⁷, Aib³⁴ ]hPTH(1-34)NH₂ ; [Cha¹¹, Aib³⁴]hPTH(1-34)NH₂ ; [Cha²⁷, Aib¹⁶ ]hPTH(1-34)NH₂ ; [Cha²⁷, Aib³⁴]hPTH(1-34)NH₂ ; [Cha²⁸, Aib¹⁶ ]hPTH(1-34)NH₂ ; [Cha²⁸, Aib³⁴]hPTH(1-34)NH₂ ; [Nle³¹ ]hPTH (1-34)NH₂ ; [hArg²⁷ ]hPTH(1-34)NH₂ ;[Dap¹, Nle⁸, 18, Tyr³⁴ ]hPTH (1-34)NH₂ ; [Nle³¹ ]bPTH(1-34)NH₂ ; [Nle³¹]rPTH (1-34)NH₂ ; [hArg²⁷ ]bPTH(1-34)NH₂ ; [hArg²⁷ ]rPTH(1-34)NH₂ ;[Cha⁷, 11, Aib¹⁹, Lys³⁰ ]hPTH(1-34)NH₂ ; [Aib¹² ]hPTH(1-34)NH₂ ; [Cha²⁴,28, 31, Lys³⁰ ]hPTH(1-34)NH₂ ; [Cha²⁸, 31 ]hPTH(1-34)NH₂ ; [Cha⁷, 11,Nle⁸, 18, Aib³⁴ ]hPTH(1-34)NH₂ ; [Aib³ ]hPTH(1-34)NH₂ ; [Cha⁸ ]hPTH(1-34)NH₂ ; [Cha¹⁵ ]hPTH(1-34)NH₂ ; [Cha⁷, 11, Aib¹⁹ ]hPTH(1-34)NH₂ ;[Cha⁷, 11 Aib¹⁶ ]hPTH(1-34)NH₂ ; [Aib¹⁷ ]hPTH(1-34)NH₂ ; [Cha⁵]hPTH(1-34)NH₂ ;[Cha⁷, 11, 15 ]hPTH(1-34)NH₂ ; [Cha⁷, 11, Nle⁸, 18,Aib¹⁹, Tyr³⁴ ]hPTH(1-34)NH₂ ; [Cha⁷, 11, Nle⁸, 18, Aib¹⁹, Lys³⁰, Tyr³⁴]hPTH(1-34)NH₂ ; [Cha⁷, 11, 15 ]hPTH(1-34)NH₂ ; [Aib¹⁷ ]hPTH(1-34)NH₂ ;[Cha⁷, 11, Leu²⁷ ]hPTH(1-34)NH₂ ; [Cha⁷, 11, 15, Leu²⁷ ]hPTH(1-34)NH₂ ;[Cha⁷, 11, 27 ] hPTH(1-34)NH₂ ; [Cha⁷, 11, 15, 27 ]hPTH (1-34)NH₂ ;[Trp¹⁵ ]hPTH(1-34)NH₂ ; [Nal¹⁵ ]hPTH (1-34)NH₂ ; [Trp¹⁵, Cha²³]hPTH(1-34)NH₂ ; [Cha¹⁵, 23 ]hPTH(1-34)NH₂ ; [Phe⁷, 11 ]hPTH(1-34)NH₂ ;[Nal⁷, 11 ]hPTH(1-34)NH₂ ; [Trp⁷, 11 ]hPTH (1-34)NH₂ ; [Phe⁷, 11, 15]hPTH(1-34)NH₂ ; [Nal⁷, 11, 15 ]hPTH (1-34)NH₂ ; [Trp⁷, 11, 15]hPTH(1-34)NH₂ ; and [Tyr⁷, 11, 15 ]hPTH (1-34)NH₂.

In another aspect, this invention relates to peptides covered by thefollowing formula: ##STR2## wherein A₁ is Ala, Ser, or Dap;

A₃ is Ser or Aib;

A₅ is His, Ile, or Cha;

A₇ is Leu, Cha, Nle, β-Nal, Trp, Pal, Phe, or p-X-Phe in which X is OH,a halogen, or CH₃ ;

A₈ is Leu, Met, or Cha;

A₁₀ is Asp or Asn;

A₁₁ is Lys, Leu, Cha, Phe, or β-Nal;

A₁₂ is Gly or Aib;

A₁₄ is Ser or His;

A₁₅ is Ile, or Cha;

A₁₆ is Gln or Aib;

A₁₇ is Asp or Aib;

A₁₈ is Leu, Aib, or Cha;

A₁₉ is Arg or Aib;

A₂₂ is Phe, Glu, Aib, Acc, or Cha;

A₂₃ is Phe, Leu, Lys, Acc, or Cha;

A₂₄ is Leu, Lys, Acc, or Cha;

A₂₅ is His, Aib, or Glu;

A₂₆ is His, Aib, or Lys;

A₂₇ is Leu, Lys, Acc, or Cha;

A₂₈ is Ile, Leu, Lys, Acc, or Cha;

A₂₉ is Ala, Glu, or Aib;

A₃₀ is Glu, Cha, Aib, Acc, or Lys;

A₃₁ is Ile, Leu, Cha, Lys, Acc, or deleted;

A₃₂ is His or deleted;

A₃₃ is Thr or deleted;

A₃₄ is Ala or deleted;

each of R₁ and R₂ is, independently, H, C₁₋₁₂ alkanyl, C₇₋₂₀phenylalkyl, C₁₁₋₂₀, naphthyalkyl, C₁₋₁₂, hydroxyalkyl, C₂₋₁₂hydroxyalkenyl, C₇₋₂₀ hydroxyphenylalkyl, or C₁₁₋₂₀hydroxynaphthylalkyl; or one and only one of R₁ and R₂ is COE₁ in whichE₁ is C₁₋₁₂ alkyl, C₂₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₇₋₂₀ phenylalkyl, C₁₁₋₂₀naphthylalkyl, C₁₋₁₂ hydroxyalkyl, C₂₋₁₂ hydroxyalkenyl, C₇₋₂₀hydroxyphenylalkyl, or C₁₁₋₂₀ hydroxynaphthylalkyl; and

R₃ is OH, NH₂, C₁₋₁₂ alkoxy, or NH--Y--CH₂ --Z in which Y is a C₁₋₁₂hydrocarbon moiety and Z is H, OH, CO₂ H or CONH₂ ;

provided that (i) at least one of A₅, A₇, A₈, A₁₁, A₁₅, A₁₈, A₂₂, A₂₃,A₂₄, A₂₇, A₂₈, A₃₀, or A₃₁ is Cha, or at least one of A₃, A₁₂, A₁₆, A₁₇,A₁₈, Al₁₉, A₂₂, A₂₅, A₂₆, A₂₉, A₃₀, or A₃₄ is Aib; or that (ii) at leastone of A₂₃, A₂₄, A₂₇, A₂₈, or A₃₁ is Lys; or a pharmaceuticallyacceptable salt thereof. In one embodiment, at least one of A₇ and A₁₁is Cha. In another embodiment, at least one of A₁₆ or A₁₉ is Aib.Specific examples of peptides of the just-recited formula include, butare not limited to, [Cha⁷ ]hPTHrP(1-34)NH₂ ; [Cha¹¹ ]hPTHrP(1-34)NH₂ ;[Cha⁷, 11 ]hPTHrP(1-34)NH₂ ; [Aib¹⁶, Tyr³⁴ hPTHrP(1-34)NH₂ ; [Aib¹⁹]hPTHrP(1-34)NH₂ ; [Aib¹⁶, 19 ]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Aib¹⁶hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Aib¹⁹ ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28,31, Glu²⁵, 29, LyS²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28,31, Lys²⁶, 27, 30 ]hPTHrP(1-34)NH₂ ; [Cha²², 23, Glu²⁵, 29 Leu²⁸, 31,Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31, Aib²⁹, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Lys²³, 26, 30, Leu²⁸, 31 ] hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Cha³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Aib³⁰ ]hPTHrP(1-34)NH₂; [Glu²², 25, 29, Leu²³, 31, Lys²⁶, 28, 30 ]hPTHrP(1-34)NH₂ ; [Cha²²,23, 24, 27, 28, 31, Glu²⁵, 29, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 25,29, Cha²³, 24, 28, 31, Lys²⁶, 27, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29,Cha²³, 24, 27, 31, Lys²⁶, 28, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29,Lys²³, 26, 30, Cha²⁴, 27, 28, 31 ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28,31, Glu²⁵, 29, Lys²⁶, 27, 30 ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 31,Glu²⁵, 29, Lys²⁶, 28, 30 ]hPTHrP(1-34)NH₂ ; [Cha²², Lys²³, 26, 30,Glu²⁵, 29, Leu²⁸, 31 ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, 31, Glu²⁵,Lys²⁶, 30, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, 31, Glu²⁵, 29,Lys²⁶, Aib³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31, Lys²⁶, 27,30, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Lys²³, 26, 30, Leu²⁸, 31, Aib²⁹]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 31, Lys 26, 28, 30, Aib²⁹]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, 22, Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 27,30 ]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, 22, 23, Glu²⁵, 29, Leu²⁸, 31, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²² 25, 29, Lys²³, 26, 30,Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²², 25, 29, Leu²³, 31, Lys²⁶, 28, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²², 25, Leu²³, 28, 31, Aib²⁹, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 11, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Aib³⁰]hPTHrP(1-34)NH₂ ; [Cha¹⁵ ₁, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, 22, Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 27, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, 22, 23, Glu²⁵, 29, Leu²⁸, 31, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, Glu²², 25, Leu²³, 28, 31, Aib²⁹, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, Glu²², 25, 29, Lys²³, 26, 30, Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Cha¹⁵, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Aib³⁰ ]hPTHrP(1-34)NH₂ ; [Cha¹⁵, Glu²², 28, 29, Leu²³, 31, Lys²⁶, 28, 30]hPTHrP(1-34)NH₂ ; [Cha¹⁵, 30, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, 22, Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶ 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 27, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, 22, 23, Glu²⁵, 29, Leu²⁸, 31, Lys²⁶, 30]hPTHrP (1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶,30]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, Leu²³, 28, 31, Aib²⁹, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, 29, Lys²³, 26, 30, Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Aib³⁰] hPTHrP(1-34)NH₂ ; [Cha⁷, 8, Glu²², 25, 29, Leu²³, 31, Lys²⁶, 28, 30]hPTHrP(1-34)NH₂ ; [Cha⁷, 8, 30, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11, 22, Met⁸, Asn₁₀, His¹⁴, Leu²³,28, 31, Glu²⁵, 29, Lys²⁶, 30 ]hPTHrP (1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11,Met⁸, Asn¹⁰, His¹⁴, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 27, 30]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11, Met⁸, Asn¹⁰, His¹⁴, Glu²², 25,29, Leu²³, 31, Lys²⁶, 28, 30 ]hPTHrP(1-34)NH₂ ; Ser¹, Ile⁵, Cha⁷, 11,Met⁸, Asn¹⁰, His¹⁴, Glu²², 25, 29, Lys²³, 26, 30, Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11, Met⁸, Asn¹⁰, His¹⁴, Glu²², 25,Leu²³, 28, 31, Aib²⁹, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷,11, Met⁸, Asn¹⁰, His¹⁴, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Aib³⁰ ]PTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11, 22, 23, Met⁸, Asn¹⁰, His¹⁴,Glu²⁵, 29, Leu²⁸, 31, Lys²⁶, 30 ]hPTHrP(1-34) NH₂ ; [Ser¹, Ile⁵, Cha⁷,11, 15, Met⁸, Asn¹⁰, His¹⁴ ]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸, Asn¹⁰,Leu¹¹, 28, 31, His¹⁴, Aib¹⁶ ]hPTHrP (1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸,Asn¹⁰, Leu¹¹, 28, 31, His¹⁴, Cha²², 23, Glu²⁵, 29, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 11, Met⁸, Asn¹⁰, His¹⁴, Glu²², 25, 29,Leu²³, 28, 31, Lys²⁶, 30 ]hPTHrP (1-34)NH₂ ; [Ser¹, Ile⁵, 7, Met⁸, Asn²¹⁰, His¹⁴, Cha¹⁵, Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Cha⁷, 8, Asn¹⁰, His¹⁴, Glu²², 25, 29, Leu²³,28, 31, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ;[Glu²², 25, 29, Leu²³, 28, 31,Lys²⁴, 26, 30 ]hPTHrP(1-34)NH₂ ; [Aib²², Leu²³, 28, 31, Glu²⁵, 29,Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Aib²⁵, Lys²⁶, 30]hPTHrP (1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, 31, Aib²⁶, Lys³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, Lys²⁶, 30, 31 ] hPTHrP (1-34)NH₂; [Ser¹, Ile⁵, Met⁸, Asn¹⁰, Leu¹¹, 23, 28, 31, His¹⁴, Cha²², Glu²⁵, 29,Lys²⁶, 30 ] hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸, Asn¹⁰, Leu¹¹, 28, 31,His¹⁴, Glu²², 25, 29, Lys²³, 26, 30 ]PTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸,Asn¹⁰, Leu¹¹, 23, 28, 31, His¹⁴, Glu²², 25, 29, Lys²⁶, 27, 30 ] hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸, Asn¹⁰, Leu¹¹, 23, 31, His¹⁴, Glu²², 25,29, Lys²⁶, 28, 30 ] hPTHrP(1-34)NH₂ ; [Ser¹, Ile⁵, Met⁸, Asn¹⁰, Leu¹¹,23, 28, 31, His¹⁴, Glu²², 25, Aib²⁹, Lys²⁶, 30 ] hPTHrP (1-34)NH₂ ;[Ser¹, Ile⁵, Met⁸, Asn¹⁰, Leu¹¹, 23, 28, 31, His¹⁴, Glu²², 25, 29,Lys²⁶, Aib³⁰ ]hPTHrP(1-34)NH₂ ; or [Ser¹, Ile⁵, Met⁸ ]hPTHrP(1-34)NH₂ ;[Glu²², 25, Ahc²³, Lys²⁶, 30, Leu²⁸, 31, Aib²⁹ ]hPTHrP(1-34)NH₂ ;[Glu²², 25, Leu²³, 28, 31, Lys²⁶, 30, Ahc²⁷, Aib²⁹ ]hPTHrP(1-34)NH₂ ;[Glu²², 25, Leu²³, 28, Lys²⁶, 30, Aib²⁹, Ahc³¹ ]hPTHrP(1-34)NH₂ ;[Glu²², 25, Cha²³, Lys²⁶, 30, Leu²⁸, 31, Aib²⁹ ]hPTHrP(1-34)NH₂ ;[Glu²², 25, Cha²³, Lys²⁶, 30, Leu²⁸, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Glu²²,25, Cha²³, Lys²⁶, 30, Aib²⁹ ]hPTHrP (1-34)NH₂ ; [Ahc²², Leu²³, 28, 31,Glu²⁵, Lys²⁶, 30, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31,Lys²⁶, Aib²⁹, Ahc³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Cha²³, Lys²⁶, 30,Aib²⁹, Leu³¹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31, Ahc²⁴, Lys²⁶,30, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 31, Lys²⁶, 30, Ahc²⁸,Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31, Lys²⁶, Aib²⁹, 30]hPTHrP (1-34)NH₂ ; [Aib²², 29, Leu²³, 28, 31, Glu²⁵, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, 31, Aib²⁶, 29, Lys³⁰]hPTHrP(1-34)NH₂ ; [Cha²², Ahc²³, Glu²⁵, 29, Lys²⁶, 30, Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, 31, Ahc²⁴, Glu²⁵, 29, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, 30,Ahc²⁷]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 31, Glu²⁵, 29, Lys²⁶, 30, Ahc²⁸]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, Leu²⁸, Ahc³⁰]hPTHrP(1-34)NH₂ ; [Cha²², 23, Glu²⁵, 29, Lys²⁶, 30, Leu³¹ ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, Glu²⁵, 29, Lys²⁶, 30, Ahc³¹ ]hPTHrP(1-34)NH₂ ; [Cha²², 23, Glu²⁵, 29, Lys²⁶, 30, Leu³¹ ]hPTHrP(1-34)NH₂ ;[Cha²², 23, Glu²⁵, 29, Lys²⁶, 30, Leu²⁸ ]hPTHrP(1-34)NH₂ ; [Cha²², 23,Glu²⁵, 29, Lys²⁶, 30 ] hPTHrP(1-34)NH₂ ; [Glu²², Leu²³, 28, 31, Aib²⁵,29, Lys²⁶, 30 ]hPTHrP(1-34)NH₂ ; [Glu²², 29, Ahc²³, Aib²⁵, Lys²⁶, 30,Leu²⁸, 31 ]hPTHrP(1-34)NH₂ ; [Ahc²², Leu²³, 28, 31, Aib²⁵, Lys²⁶, 30,Glu²⁹ ] hPTHrP(1-34)NH₂ ; [Aib²², 25, Leu²³, 28, 31, Lys²⁶, 30, Glu²⁹]hPTHrP (1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Ahc²⁴, Aib²⁵, Lys²⁶, 30]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Aib²⁵, 26, Lys³⁰]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Aib²⁵, Lys²⁶, 30, Ahc²⁷ ]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 31, Aib²⁵, Lys²⁶, 30, Ahc²⁸]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, Aib²⁵, Lys²⁶, 30, Ahc³¹]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Aib²⁵, 30, Lys²⁶]hPTHrP(1-34)NH₂ ; [Glu²², 29, Leu²³, 28, 31, Aib²⁵, Lys²⁶, Ahc³⁰]hPTHrP(1-34)NH₂ ; [Glu²², 29, Cha²³, Aib²⁵, Lys²⁶, 30, Leu²⁸, 31]hPTHrP(1-34))NH₂ ; [Glu²², 29, Cha²³, Aib²⁵, Lys²⁶, 30, Leu²⁸, 31]hPTHrP(1-34)NH₂ ; [Glu²², 29, Cha²³, Aib²⁵, Lys²⁶, 30,]hPTHrP(1-34)NH₂; [Glu²², 29, Cha²³, Aib²⁵, Lys²⁶, 30, Leu²⁸ ]hPTHrP(1-34)NH₂ ; [Glu²²,25, 29, Cha²³, Lys²⁶, Leu²⁸, 31, Aib³⁰ ] hPTHrP (1-34)NH₂ ; [Glu²², 25,29, Cha²³, Lys²⁶, Aib³⁰, Leu³¹ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Cha²³,Lys²⁶, Aib³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Cha²³ Lys²⁶, Leu²⁸,Aib³⁰ ]hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Ahc²⁷,Aib³⁰ ] hPTHrP(1-34)NH₂ ; [Glu²², 25, 29, Leu²³, 28, 31, Ahc²⁴, Lys²⁶,Aib³⁰ ]hPTHrP (1-34)NH₂ ; [Ahc²², Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, Aib³⁰]hPTHrP(1-34)NH₂ ; [Aib²², 30, Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶]hPTHrP(1-34)NH₂ ; [Glu²², 25, Leu²³, 28, Lys²⁶, 30, 31, Aib²⁹ ]hPTHrP(1-34)NH₂ ; [Cha²², Leu²³, 28, Glu²⁵, 29, Lys²⁶, 30, 31 ]hPTHrP(1-34)NH₂; [Ahc²², Leu²³, 28, Glu²⁵, 29, Lys²⁶, 30, 31 ] hPTHrP(1-34)NH₂ ;[Glu²², 25, 29, Leu²³, 28, Lys²⁶, 30, 31, Ahc³⁰ ]hPTHrP (1-34)NH₂ ;[Glu²², 25, 29, Leu²³, 28, 31, Lys²⁶, Ahc³⁰ ]hPTHrP(1-34)NH₂ ; [Ahc²²,Leu²³, 28, 31, Glu²⁵, 29, Lys²⁶, 30 ]hPTHrP (1-34)NH₂ ; [Glu²², 25, 29,Leu²³, 28, Lys²⁶, 30, 31, Ahc²⁷ ]hPTHrP(1-34)NH₂.

With the exception of the N-terminal amino acid, all abbreviations (e.g.Ala or A₁) of amino acids in this disclosure stand for the structure of--NH--CH(R)--CO--, wherein R is a side chain of an amino acid (e.g., CH₃for Ala). For the N-terminal amino acid, the abbreviation stands for thestructure of ═N--CH(R)--CO--, wherein R is a side chain of an aminoacid. β-Nal, Nle, Dap, Cha, Nva, Amp, Pal, Ahc, and Aib are theabbreviations of the following α-amino acids: β-(2-naphthyl)alanine,norleucine, α,β-diaminopropionic acid, cyclohexylalanine, norvaline,4-amino-phenylalanine, 3-pyridinylalanine, α-aminocyclohexanecarboxylicacid, and α-aminoisobutyric acid, respectively. What is meant by Acc isan amino acid selected from the group of α-aminopropanecarboxylic acid;α-aminobutanecarboxylic acid; α-aminopentanecarboxylic acid;α-aminohexanecarboxylic acid; α-aminoheptanecarboxylic acid;α-aminooctanecarboxylic acid; and α-aminononanecarboxylic acid. In theabove formula, hydroxyalkyl, hydroxyphenyl-alkyl, andhydroxynaphthylalkyl may contain 1-4, hydroxy substituents. Also, COE₁stands for --C═O·E₁. Examples of --C═O·E₁ include, but are not limitedto, acetyl and phenylpropionyl.

A peptide of this invention is also denoted herein by another format,e.g., [Cha⁷, 11 ]hPTH(1-34)NH₂, with the substituted amino acids fromthe natural sequence placed between the second set of brackets (e.g.,Cha⁷ for Leu⁷, and Cha¹¹ for Leu¹¹ in hPTH). The abbreviation hPTHstands for human PTH, hPTHrP for human PTHrP, rPTH for rat PTH, and bPTHfor bovine PTH. The numbers between the parentheses refer to the numberof amino acids present in the peptide (e.g., hPTH(1-34) is amino acids1, through 34 of the peptide sequence for human PTH). The sequences forhPTH(1-34), hPTHrP(1-34), bPTH(1-34), and rPTH(1-34) are listed inNissenson, et al., Receptor, 3:193 (1993). The designation "NH₂ " inPTH(1-34)NH₂ indicates that the C-terminus of the peptide is amidated.PTH(1-34), on the other hand, has a free acid C-terminus.

Each of the peptides of the invention is capable of stimulating thegrowth of bone in a subject (i.e., a mammal such as a human patient).Thus, it is useful in the treatment of osteoporosis and bone fractureswhen administered alone or concurrently with antiresorptive therapy,e.g., bisphosphonates and calcitonin.

The peptides of this invention can be provided in the form ofpharmaceutically acceptable salts. Examples of such salts include, butare not limited to, those formed with organic acids (e.g., acetic,lactic, maleic, citric, malic, ascorbic, succinic, benzoic,methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids(e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), andpolymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic,polyglycolic, or copolymers of polylactic-glycolic acids).

A therapeutically effective amount of a peptide of this invention and apharmaceutically acceptable carrier substance (e.g., magnesiumcarbonate, lactose, or a phospholipid with which the therapeuticcompound can form a micelle) together form a therapeutic composition(e.g., a pill, tablet, capsule, or liquid) for administration (e.g.,orally, intravenously, transdermally, pulmonarily, vaginally,subcutaneously, nasally, iontophoretically, or by intratracheally) to asubject. The pill, tablet, or capsule that is to be administered orallycan be coated with a substance for protecting the active compositionfrom the gastric acid or intestinal enzymes in the stomach for a periodof time sufficient to allow it to pass undigested into the smallintestine. The therapeutic composition can also be in the form of abiodegradable or nonbiodegradable sustained release formulation forsubcutaneous or intramuscular administration. See, e.g., U.S. Pat. No.3,773,919 and 4,767,628 and PCT Application No. WO 94/15587. Continuousadministration can also be achieved using an implantable or externalpump (e.g., INFUSAID™ pump). The administration can also be conductedintermittently, e.g., single daily injection, or continuously at a lowdose, e.g., sustained release formulation.

The dose of a peptide of the present invention for treating theabove-mentioned diseases or disorders varies depending upon the mannerof administration, the age and the body weight of the subject, and thecondition of the subject to be treated, and ultimately will be decidedby the attending physician or veterinarian.

Also contemplated within the scope of this invention is a peptidecovered by the above generic formula for use in treating diseases ordisorders associated with deficiency in bone growth or the like, e.g.,osteoporosis or fractures.

Other features and advantages of the present invention will be apparentfrom the detailed description and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Based on the description herein, the present invention can be utilizedto its fullest extent. The following specific examples are to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. Further, all publications citedherein are incorporated by reference.

Structure

PTH(1-34) has been reported to have two amphophilic alpha helicaldomains. See, e.g., Barden, et al., Biochem., 32:7126 (1992). The firstα-helix is formed between amino acid residues 4 through 13, while thesecond α-helix is formed between amino acid residues 21 through 29. Somepeptides of this invention contain the substitution of Cha for one ormore residues within or near these two regions of PTH(1-34), e.g., Cha⁷and Cha¹¹ within the first α-helix or Cha²⁷ and Cha²⁸ within the secondα-helix.

Also covered by this invention are variants of PTH(1-34) with thesubstitution of Aib for a residue adjacent to the α-helixes, e.g.,Aib¹⁶, Aib¹⁹, and Aib³⁴ ; hArg²⁷ and Nle³¹, or the substitution of Dpafor the N-terminal residue.

Synthesis

The peptides of the invention can be prepared by standard solid phasesynthesis. See, e.g., Stewart, J. M., et al., Solid Phase Synthesis(Pierce Chemical Co., 2d ed. 1984). The following is a description ofhow [Aib³⁴ ]hPTH (1-34)NH₂ was prepared. Other peptides of the inventioncan be prepared in an analogous manner by a person of ordinary skill inthe art.

The peptide was synthesized on an Applied Biosystems (Foster City,Calif.) model 430A peptide synthesizer which was modified to doaccelerated Boc-chemistry solid phase peptide synthesis. See Schnoize,et al., Int. J. Peptide Protein Res., 90:180 (1992).4-Methylbenz-hydrylamine (MBHA) resin (Peninsula, Belmont, Calif.) withthe substitution of 0.93 mmol/g was used. The Boc amino acids (Bachem,Calif., Torrance, Calif.; Nova Biochem., Lajolla, Calif.) were used withthe following side chain protection: Boc-Arg(Tos)--OH,Boc-Asp(OcHxl)--OH, Boc-Asn(Xan)--OH, Boc-Glu(OcHxl)--OH,Boc-His(DNP)--OH, Boc-Asn-GH, Boc-Val-OH, Boc-Leu-OH, Boc-Ser-OH,Boc-Gly-OH, Boc-Met-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(2ClZ)--OH,Boc-Ser(Bzl)--OH, and Boc-Trp(Fm)--OH. The synthesis was carried out ona 0.14 mmol scale. The Boc groups were removed by treatment with 100%TFA for 2×1 min. Boc amino acids (2.5 mmol) were pre-activated with HBTU(2.0 mmol) and DIEA (1.0 mL) in 4 mL of DMF and were coupled withoutprior neutralization of the peptide-resin TFA salt. Coupling times were5 min except for the Boc-Aib-OH and the following residue,Boc-Asn(Xan)--OH, wherein the coupling times were 20 min.

At the end of the assembly of the peptide chain, the resin was treatedwith a solution of 20% mercaptoethanol/10% DIEA in DMF for 2×30 min. toremove the DNP group on the His side chain. The N-terminal Boc group wasthen removed by treatment with 100% TFA for 2×2 min. Afterneutralization of the peptide-resin with 10% DIEA in DMF (1×1 min.), theformyl group on the side chain of Trp was removed by treatment with asolution of 15% ethanolamine/15% water/70% DMF for 2×30 min. Thepartially-deprotected peptide-resin was washed with DMF and DCM anddried under reduced pressure. The final cleavage was done by stirringthe peptide-resin in 10 mL of HF containing 1 mL of anisole at 0°C. for75 min. HF was removed by a flow of nitrogen. The residue was washedwith ether (6×10 mL) and extracted with 4N HOAc (6×10 mL).

The peptide mixture in the aqueous extract was purified on areversed-phase preparative high pressure liquid chromatography (HPLC)using a reversed phase VYDAC C₁₈ column (Nest Group, Southborough,Mass.). The column was eluted with a linear gradient (10% to 45% ofsolution B over 130 min.) at a flow rate of 10 mL/min (Solution A=0.1%aqueous TFA; Solution B=acetonitile containing 0.1% of TFA). Fractionswere collected and checked on analytical HPLC. Those containing pureproduct were combined and lyophilized to dryness. 62.3 mg of a whitesolid was obtained. Purity was >99% based on analytical HPLC analysis.Electro-spray mass spectrometer analysis gave the molecular weight at4054.7 (in agreement with the calculated molecular weight of 4054.7).

The synthesis and purification of [Cha⁷, 11 ]hPTH (1-34)NH₂ was carriedout in the same manner as the above synthesis of [Aib³⁴ ]hPTH(1-34)NH₂.The protected amino acid Boc-Cha-OH was purchased from Bachem, Calif.The purity of the final product was >98%, and the electron-spray massspectrometer gave the molecular weight at 4197.0 (calculated molecularweight is 4196.9).

The full names for the abbreviations used above are as follows: Boc fort-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan forxanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMFfor dimethylformamide, DCM for dichloromethane, HBTU for2-(1H-Benzotriazo1-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate, DIEA for diisopropylethylamine, HOAc for aceticacid, TFA for trifluoroacetic acid, 2ClZ for 2-chlorobenzyloxycarbonyland OcHxl for O-cyclohexyl.

The substituents R₁ and R₂ of the above generic formula may be attachedto the free amine of the N-terminal amino acid by standard methods knownin the art. For example, alkyl groups, e.g., C₁₋₁₂ alkyl, may beattached using reductive alkylation. Hydroxyalkyl groups, e.g., C₁₋₁₂hydroxyalkyl, may also be attached using reductive alkylation whereinthe free hydroxy group is protected with a t-butyl ester. Acyl groups,e.g., COE₁, may be attached by coupling the free acid, e.g., E₁ COOH, tothe free amine of the N-terminal amino acid by mixing the completedresin with 3 molar equivalents of both the free acid anddiisopropylcarbodiimide in methylene chloride for one hour and cyclingthe resulting resin through steps (a) to (f) in the above wash program.If the free acid contains a free hydroxy group, e.g.,p-hydroxyphenylpropionic acid, then the coupling should be performedwith an additional 3 molar equivalents of HOBT.

Other peptides of this invention can be prepared in an analogous mannerby a person of ordinary skill in the art.

Functional Assays

A. Binding to PTH Receptor

The peptides of the invention were tested for their ability to bind tothe PTH receptor present on SaOS-2 (human osteosarcoma cells). SaOS-2cells (American Type Culture Collection, Rockville, Md.; ATCC #HTB 85)were maintained in RPMI 1640 medium (Sigma, St. Louis, Mo.) supplementedwith 10% fetal bovine serum (FBS) and 2 mM glutamine at 37° C. in ahumidified atmosphere of 5% CO₂ in air. The medium was changed everythree or four days, and the cells were subcultured every week bytrypsinization.

SaOS-2 cells were maintained for four days until they had reachedconfluence. The medium was replaced with 5% FBS in RPMI 1640 medium andincubated for 2 hrs at room temperature with 10×10⁴ cpm mono-¹²⁵I-[Nle⁸,18, Tyr³⁴ (3-¹²⁵ I)]bPTH(1-34)NH₂ in the presence of a competingpeptides of the invention at various concentrations between 10⁻¹¹ M to10⁻⁴ M. The cells were washed four times with ice-cold PBS and lysedwith 0.1 M NaOH, and the radioactivity associated with the cells wascounted in a scintillation counter. Synthesis of mono-¹²⁵ I-[Nle⁸,18,Tyr³⁴ (3-¹²⁵ I)] bPTH(1-34)NH₂ was carried out as described in Goldman,M. E., et al., Endocrinol., 123:1468 (1988).

The binding assay was conducted with various peptides of the invention,and the IC₅₀ value, (half maximal inhibition of binding of mono-¹²⁵I-[Nle⁸,18, Tyr³⁴ (3-¹²⁵ I)]bPTH(1-34)NH₂, for each peptide wascalculated.

As shown in Table I, all of the tested peptides had a high bindingaffinity for the PTH receptor on the SaOS-2 cell.

B. Stimulation of Adenylate Cyclase Activity

The ability of the peptides of the invention to induce a biologicalresponse in SaOS-2 cells were measured. More specifically, anystimulation of the adenylate cyclase was determined by measuring thelevel of synthesis of cAMP (adenosine 3', 5'-monophosphate) as describedpreviously in Rodan, et al., J. Clin. Invest. 72: 1511 (1983) andGoldman, et al., Endocrinol., 123:1468 (1988). Confluent SAOS-2 cells in24 wells plates were incubated with 0.5 μCi [³ H]adenine (26.9 Ci/mmol,New England Nuclear, Boston, Mass.) in fresh medium at 37° C. for 2 hrs,and washed twice with Hank's balanced salt solution (Gibco,Gaithersburg, Md.). The cells were treated with 1 mM IBMX[isobutylmethyl-xanthine, Sigma, St. Louis, Mo.] in fresh medium for 15min, and the peptides of the invention were added to the medium toincubate for 5 min. The reaction was stopped by the addition of 1.2 Mtrichloroacetic acid (TCA) (Sigma, St. Louis, Mo.) followed by sampleneutralization with 4 N KOH. cAMP was isolated by the two-columnchromatographic method (Salmon, et al., 1974, Anal. Biochem. 58, 541).The radioactivity was counted in a scintillation counter (LiquidScintillation Counter 2200CA, PACKARD, Downers Grove, Ill.).

The respective EC₅₀ values (half maximal stimulation of adenylatecyclase) for the tested peptides were calculated and shown in Table I.All tested peptides were found to be potent stimulators of adenylatecyclase activity, which is a biochemical pathway indicative as aproximal signal for osteoblast proliferation (e.g., bone growth).

                  TABLE I                                                         ______________________________________                                                                 Kd      EC.sub.50                                                                      PEPTIDE (μM) (nM)                        ______________________________________                                        [Cha.sup.7,11 ]hPTH(1-34)NH.sub.2                                                                      0.01    0.6                                            [Cha.sup.23 ]hPTH(1-34)NH.sub.2 0.2 20                                        [Cha.sup.24 ]hPTH(1-34)NH.sub.2 0.1 10                                        [Nle.sup.8,18,Cba.sup.27 ]hPTH(1-34)NH.sub.2; 0.05 2                          [Cha.sup.28 ]hPTH(1-34)NH.sub.2 0.05 2.5                                      [Cha.sup.31 ]hPTH(1-34)NH.sub.2 0.03 4                                        [Aib.sup.16 ]hPTH(1-34)NH.sub.2; 0.004 0.7                                    [Aib.sup.19 ]hPTH(1-34)NH.sub.2; 0.005 0.6                                    [Aib.sup.34 ]hPTH(1-34)NH.sub.2; 0.007 3                                      [Nle.sup.31 ]hPTH(1-34)NH.sub.2; 0.004 0.7                                    [hArg.sup.27 ]hPTH(1-34)NH.sub.2 0.007 1                                      [Dap, Nle.sup.8,18,Tyr.sup.34 ]hPTH(1-34)NH.sub.2 0.150 10                    [Cha.sup.24,28,31,Lys.sup.30 ]bPTH(1-34)NH.sub.2; 0.5 7                       [Cha.sup.7,11,Nle.sup.8,18,Tyr.sup.34 ]hPTH(1-34)NH.sub.2 0.006 0.6                                           [Cha.sup.7,11,Nle.sup.8,18,Aib.sup.16,19                                     ,Tyr.sup.34 ]hPTH(1-34)NH.sub.2 0.005                                         1.5                                            [Cha.sup.7,11,Nle.sup.8,18,31, Aib.sup.16,19,Tyr.sup.34 ]hPTH(1-34)NH.su                                     b.2 0.04 4                                     [Cha.sup.11 ]hPTH(1-34)NH.sub.2 0.005 2                                       [Cha.sup.28,31 ]hPTH(1-34)NH.sub.2 0.06 7                                     [Cha.sup.7,11,Nle.sup.8,18,Aib.sup.34 ]hPTH(1-34)NH.sub.2 0.03 1.5                                            [Cha.sup.15 ]hPTH(1-34)NH.sub.2 0.005                                        1.3                                            [Cha.sup.7,11,Aib.sup.19 ]hPTH(1-34)NH.sub.2 0.007 0.5                        [Cha.sup.7,11,Aib.sup.16 ]hPTH(1-34)NH.sub.2 0.004 1.1                        [Aib.sup.16,19 ]hPTH(1-34)NH.sub.2 0.004 0.6                                  [Aib.sup.12 ]hPTH(1-34)NH.sub.2 0.005 2                                       [Aib.sup.3 ]hPTH(1-34)NH.sub.2 0.004 1.1                                      [Cha.sup.7,11,Aib.sup.19,Lys.sup.30 ]hPTH(1-34)NH.sub.2 0.004 2                                               [Cha.sup.7 ]hPTH(1-34)NH.sub.2 0.02 2.3       [Cha.sup.24,28,31 ]hPTH(1-34)NH.sub.2 1.0 30                                  [Aib.sup.17 ]hPTH(1-34) 0.05 3                                                [Cha.sup.7,11,15 ]hPTH(1-34) 0.01 1.4                                       ______________________________________                                    

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the claims.

What is claimed is:
 1. The compound of the formula [Glu²²,25,Leu²³,28,31, Aib²⁹, Lys²⁶,30 ]hPTHrP(1-34)NH₂.